The objective of this work is to investigate the role of HIV-1 and other retroviral Gag and nucleocapsid proteins (NCs) in the regulation of processes involving nucleic acid structural transitions and viral self assembly in retroviral systems. The PI has pioneered the development of new single molecule nucleic acid (NA) stretching methods that can be used to quantitatively probe nucleic acid structural rearrangements and protein-nucleic acid interactions. The proposed work continues to develop novel methods to test specific hypotheses concerning nucleic acid-protein interactions that are important for retroviral packaging and replication. The results of these studies are integrated with collaborative in vivo work. The capability of NCs to rearrange nucleic acids to facilitate reverse transcription, referred to as NA chaperone acitivity, is essential for retroviral replication. NA stretching methods are uniquely well suited for probing NA chaperone activity because nucleic acid structural rearrangement and packaging can be directly controlled and the resulting response can be quantified. A new method will be developed to mechanically probe reverse transcription and characterize the effects of proteins important for HIV-1 replication on this process. The specific aims are: (1) To quantify the interactions of wild type and mutant HIV-1 and other retroviral nucleic acid chaperone proteins with single- and double-stranded DNA and specific RNA structures. This work will test the hypothesis that nucleic acid chaperone activity is comprised of three critical elements: nucleic acid aggregation, nucleic acid destabilization, and rapid protein-nucleic acid interactions kinetics. (2) To characterize the DNA interactions of APOBEC proteins that may interfere with reverse transcription. We hypothesize that APOBEC proteins inhibit reverse transcription by altering specific DNA interactions. To test this hypothesis, will examine the thermodynamics and kinetics of human APOBEC interactions with single- and double-stranded DNA. (3) To quantify the nucleic acid interactions of wild type and mutant HIV-1 Gag. Although HIV-1 NC is the domain of Gag that is primarily responsible for its interactions with NAs, these proteins facilitate significantly different processes in the cell. This aim seeks to understand how NC and Gag exhibit different NA interactions and how these interactions alter reverse transcription and the NA packaging process. (4) To mechanically measure reverse transcriptase (RT) polymerization in the presence of NC and APOBEC3G and determine how they alter RT's essential processes. PUBLIC HEALTH RELEVANCE: Nucleic acid packaging and reverse transcription are critical components of retroviral replication. This project develops novel methods for characterizing these processes to contribute towards development of a molecular level understanding of retroviral replication, which in turn will help to develop drugs that target these processes.